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  • 1. IMMUNOASSAY DEVELOPMENT P A R T 5
  • 2. 381© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/B978-0-08-097037-0.00025-7 Immunoassay has become one of the commonest method- ologies in clinical diagnostics and life-science research. Enzyme-linked immunosorbent assay (ELISA) is the most widely used immunoassay configuration, and the ELISA acronym is often used as a euphemism for immunoassays of all types. By strict definition, it encompasses any anti- body- or antigen-coated solid-phase immunoassay with an enzyme involved in the signal generation process. Some commercially developed immunoassay systems have moved away from the use of enzymes in signal generation, but most immunoassays are still ELISAs. The principle of ELISA is very sound. Since a single molecule of enzyme can convert many molecules of sub- strate, the signal generation is highly amplified. Typically ELISAs are performed using microtiter® polystyrene plates. Laboratory-scale plate incubators, washers, and spectrophotometers provide a flexible and cheap platform for applying the sensitivity and specificity of immunoas- says to an almost limitless range of analytes. The ability of in-house developed ELISAs to provide cheap, reliable tests for screening large numbers of sam- ples has been an irresistible attraction to countless researchers and clinical biochemists working on limited budgets. Coaxing the best possible performance out of two bio- logical entities (antibodies and enzymes) can be demand- ing at times, and there are traps for the unwary. But the effort is rewarded by the ability to test thousands of sam- ples quickly and cheaply for minute concentrations of analyte. Fortunately, a huge range of antibodies is commercially available, and the enzymes in common use are well charac- terized and have proved reliable in countless situations. ELISA is used for the detection and quantification of antigens, antibodies, hormones, and other molecules. Besides applications in clinical diagnostics, ELISA is used as the exclusive assay method for many research purposes, such as characterizing new proteins and developing new drug therapies. Although many in vitro diagnostics compa- nies manufacture reagent kits for research use, they cannot keep pace with the ever-expanding needs of researchers investigating new proteins in the proteome era. Some manufacturers provide customized services for research- ers, but the specific research requirements are often beyond the capabilities of the assays provided. Many research-oriented ELISA kits are expensive and unreli- able. Therefore, researchers often develop in-house ELISA methods. Developing an ELISA method used to be a remarkably complex task, demanding multidisciplinary knowledge and skills. But in the past decade, technologies involved in ELISA have improved significantly, although the basic principles stay the same. The intent of this chapter is to provide general guidelines for researchers to develop their own ELISA methods, tailored to their requirements, by focusing on the basic concepts and best practice for assay design for in-house use. Elsewhere in this book, there is more detail on many of the topics covered here, but this chapter describes a practical approach for assay development. The basic ELISA procedure involves plate coating, blocking, washing, and signal generation and measure- ment. ELISA is usually performed in 96-well plates or 8- or 12-well strips made from polystyrene, which will pas- sively bind proteins. In ELISA, antibodies are commonly bound to the polystyrene directly unless antigens are bound to detect antibodies in the sample. The residual, uncoated area of each well is then saturated with an inert protein (such as BSA) to prevent any nonspecific binding of the reagents. Next, the samples are added to the wells and incubated so that the analyte (antigen or antibody from samples) binds to the coated antibodies. It is the immobilization of reagents on the microplate surface and subsequent binding of analyte that makes ELISAs rela- tively easy to perform. Washing the wells removes nonspe- cifically bound materials, allowing ELISAs to measure specific analytes within a crude preparation. Finally, the reaction is quantified with a specific enzyme-conjugated antibody, which, together with a substrate, induces a pro- portional change in a color reaction. The buildup of layers as the assay progresses, with the analyte held between the immobilized and enzyme-conjugated antibodies, is the reason why ELISAs are colloquially known as sandwich assays. See Fig. 1. To design and develop an ELISA method to meet ana- lytical performance requirements, researchers should be familiar with the concepts and stages of the development process. In practice, the stages of developing a particular ELISA for in-house use are G Development. G Optimization. G Validation. At the development stage, the key elements of the assay design are selected, such as antigens and antibodies, the formulation of the reagents for the immunoreaction and the signal generation system (enzyme, antibody, and method of detection). At the optimization stage, the key factors are tested and titrated to maximize performance and achieve the best possible trade-offs, for example, between convenience and analytical capability. Finally, at the validation stage, the method is verified for the required performance. Developing an ELISA method can be very short or quite long, depending on the availability and qual- ity of the key raw materials and the technical challenges encountered with it. In a well-organized laboratory, with suitable antibodies available, an expert immunoassay spe- cialist can sometimes develop an acceptable ELISA in a few days. But usually, an ELISA takes several months or even years to develop. Practical Guide to ELISA Development Jianwen He (he.jianwen@yahoo.com) C H A P T E R 5.1
  • 3. 382 The Immunoassay Handbook FIGURE 1 Example of immunometric format ELISA.
  • 4. 383CHAPTER 5.1 Practical Guide to ELISA Development Fundamental Requirements The ideal ELISA developed in-house should be G Sensitive: the method is capable of measuring the ana- lyte at a sufficiently low concentration for the intended application of the test. G Specific: the method has negligible cross-reactivity to molecules structurally similar to the analyte that may be present in the samples. G Simple: the method is easy to perform and gives quick results. G Stable: the reagents used in the assay are thermostable, and the analytical performance is robust. G Safe: the reagent components used in the method are not harmful and require no specific handling. Key Steps of ELISA Development ESTABLISH THE REQUIREMENTS The first stage of any design process is the definition of the requirements, and for ELISA, this includes the technical constraints, raw materials, and analytical performance expectations. The requirements must answer the following questions: G What is to be measured (analyte)? G What are the sample matrices (serum, plasma, cell lysates, ascites, etc.)? G What is the detection limit required? G How specific must the ELISA be? G What is the measurement range? G How accurate must the ELISA be? G What is the detection mode (colorimetric, fluorescence, or chemiluminescence) and which plate reader(s) can be used? G What are the requirements for stability of the reagents, standards, calibrators, controls, and samples? Based on the answers to the questions above, a prototype ELISA method is first developed to establish the proof of concept, i.e., the feasibility of the project. Optimization of the component formulations and protocol steps comes later. UNDERSTAND THE ANALYTE The analyte is the target that the ELISA needs to detect and in most cases quantify. Common analytes are proteins (including antibodies), smaller naturally occurring sub- stances such as steroid hormones, and drugs and other syn- thetic compounds. It is particularly important to understand the characteristics of the analyte in the context of ELISA method development. The characteristics of analytes can be found through literature and patent reviews. Such information includes structure, molecular weights, isoelectric point (pI) value, antigenicity, solubil- ity, and thermal stability. Research the behavior of the analyte in the type of sam- ple under test. For example in blood, check if the analyte is bound to a carrier protein, such as albumin. Two important considerations for ELISA are the immunogenicity of the analyte: its ability to provoke an antibody response; and antigenicity: its ability to bind to antibodies. These are two different but related characteris- tics. This indicates how difficult it will be to acquire or generate suitable antibodies. Understanding the size and the spatial relationship of the epitopes (antigenic determi- nants) on the analyte is very useful in the selection of anti- bodies and can greatly affect assay performance. Linear epitopes consist of a continuous sequence of amino acids in protein analytes, and conformational epitopes exist when discontinuous sections of amino acids form an anti- genic determinant. The distribution and variability of epi- topes are also informative for ELISA reagent formulation. When ELISAs are required to detect specific antibodies, it is necessary to define the class or subtypes of the antibody to be measured. Sometimes, ELISA is used to measure total antibodies. Small molecules (called haptens) can elicit an immune response only when attached to a large carrier, usually a protein, although the carrier may be one that by itself does not elicit an immune response. Once antibodies have been generated, the small-molecule hapten may also be able to bind to the antibody. For research applications, the analyte to be measured can be a recombinant form of the natural protein. What is the difference in antigenicity, stability, binding affinity, and reactivity between the recombinant protein and its natural counterpart? The expected analyte concentration range likely to be encountered in the sample matrix of choice determines the detection limit and the measurable range that should be achieved in a validated method. CHOOSE A SUITABLE ASSAY FORMAT Four formats are frequently used for ELISA: immunomet- ric (sandwich), competitive, indirect, and immunocapture. The selection of ELISA format depends on the availability of key reagents, and the assay sensitivity and dynamic range required for the application. Immunometric The immunometric (sandwich) format usually uses two antibodies that preferably bind to different sites on the antigen. The primary antibody, which is highly specific for the antigen, is attached to the microtiter plates. The samples containing the analyte are then added, followed by addition of the detection antibody, which is conju- gated to an enzyme. As a result the analyte is “sand- wiched” between the two antibodies. Sometimes, to increase sensitivity, multiple antibodies can be attached to the microtiter plates for capture. In the final step of the assay, the signal generated is proportional to the amount of target analyte present in the sample. Com- pared to other formats, the immunometric format is more sensitive, precise, and robust. Not surprisingly therefore, it is the most commonly used. However, the analyte molecule needs to be large enough (with a molec- ular weight greater than 6000Da) to have two separate antigenic sites (see Fig. 1).
  • 5. 384 The Immunoassay Handbook Competitive The competitive format is used for analytes with a low molecular weight. It relies on a single antibody specific for the analyte. For optimal results, affinity purified antibodies are preferred. Compared to the immunometric format, the sensitivity of competitive assay is more defined by the equilibrium constant of the antibodies, precision of signal measurement, and level of nonspecific binding. Develop- ment and validation of a competitive ELISA require con- siderable expertise in reagent characterization and method development. In general, a competitive ELISA is not as sensitive as a sandwich ELISA, has a narrower working concentration range, and is more susceptible to matrix effects. The timing of the various incubation steps is more critical in the design of a competitive ELISA. See Fig. 2. Indirect The indirect format is used for detection of specific anti- bodies, e.g., antiviral IgG. The format uses antigen coated onto the plate to capture antibodies, and then, the captured antibodies are detected by species-specific anti-IgG or IgM. The indirect format is also susceptible to nonspe- cific binding. The purity and specificity of the antigen to be coated on the microtiter plates are very critical to the specificity of the ELISA method. See Fig. 3. Immunocapture The immunocapture format is also designed for detection of specific antibodies, usually IgM antibodies. This format uses animal anti-IgM to capture the IgM in the sample, then a specific enzyme-labeled antigen or antigen paired with enzyme-labeled specific antibody is used to detect the IgM of interest. This format requires a specific antigen with high purity for labeling or bridging to the enzyme- labeled antibody. See Fig. 4. Solid Phase For research purposes, the ELISA method is usually het- erogeneous and requires a solid phase that facilitates sepa- rating the bound from the unbound analyte and matrices. Immobilizing or coating is the process used to attach the specific antibodies or antigens onto the solid phase that FIGURE 2 Competitive format.
  • 6. 385CHAPTER 5.1 Practical Guide to ELISA Development captures analytes in the later steps. Microtiter plates, which are easy to handle and process, are the usual solid phase for ELISA. They are available either in the form of 96-well plates or 8- or 12-well strips. Due to its high capacity for protein binding, polystyrene is the usual plastic chosen. Flat-bottomed wells are recommended for spectropho- tometer readings, round-bottomed (U-shaped) wells are more suitable for visual assessment. ANTIBODY OR ANTIGEN REAGENTS Affinity Affinity is the foundation of all immunoassay develop- ment, including ELISA. In general, the binding process between antigen and antibody can be attributed to ener- getic factors, such as van der Waals forces, hydrogen bonds, and ion pairs. But the intrinsic affinity for anti- body–antigen binding is affected by many factors, includ- ing temperature, time, pH, ionic strength, detergent type and concentration, and the concentration of other macromolecules. Typically, the goal in the feasibility phase of ELISA development is to achieve high assay sensitivity by selecting workable antibody pairs and by maximizing the antibody-antigen binding affinity. Ulti- mately, in this phase, we want to identify the best anti- body or antibody pair and create an environment in which the antibody and antigen are most adapted to each other. FIGURE 3 Indirect format.
  • 7. 386 The Immunoassay Handbook Antibodies and antigens are key biologicals in ELISA design. For in-house ELISA development, the antibodies can be from donation, purchased, or generated by the researchers. Many kinds of antibodies can be used in ELISA, but monoclonal and polyclonal IgG antibodies are the most common. Affinity and specificity are the key characteristics for antibody selection. Antibody affinity is determined by comparing the rate of the antibody–antigen formation to the rate of dissociation. Often, the affinity constant deter- mines the potential sensitivity of the assay. It is recom- mended that the affinity constant be greater than 1010–1011 L/mol and ideally 1012 L/mol. Antibodies with higher affinity may be less susceptible to interferences (from the environment, instrument, or different detection tech- niques) and typically these antibodies provide more robust results. Binding affinity is influenced by the antigen: those with multiple repeating epitopes naturally generate higher avidity in binding than antigens with a single epitope. An affinity constant <108 L/mol is usually not suitable for an ELISA. Many companies do not sell their best antibody clones but retain them for their kits. This is the reason that in-house assays sometimes cannot match the performance of the commercial kits for the same analyte. To ensure detection specificity, analyte-specific anti- bodies should be used. Consult manufacturers’ data sheets for information on cross-reactivity. However, highly spe- cific antibodies may not detect all the antigen isoforms, and weak affinity for certain isoforms may result in assay drift and imprecision. To achieve the right balance, it is important to consider how the assay will be used. FIGURE 4 Immunocapture format, using a test for anti-hepatitis A virus IgM as an example.
  • 8. 387CHAPTER 5.1 Practical Guide to ELISA Development In assays in which antigen is employed as a reagent, i.e., competitive, indirect, and immunocapture assays, it is important to understand the biochemistry of the antigen, and how it behaves with respect to the antibodies it will bind with in the assay. Antibody Pair The antibody lies at the heart of an ELISA. Identifying the best antibodies and the correct roles for these antibodies (capture vs detection) is a critical balancing act. The cap- ture antibody typically has high affinity and specificity for the analyte in the sample, whereas detection antibody may contribute less to analyte specificity. This is particularly true in ELISA methods with multiple wash steps prior to the addition of the detection antibody. As discussed above, the antibody-antigen binding affinity is usually what determines ELISA sensitivity and, as a result, researchers need to prudently select antibody pairs in order to avoid any potential cross-reaction by structurally related molecules. The choice of antibody pair will depend on the antigen to be detected and the availability of antibodies to differ- ent epitopes on the antigen. A large number of monoclo- nal antibodies (mAb) and polyclonal antibodies (pAb) are available commercially and can be identified quickly by searching on websites. Alternatively, new specific antibod- ies may be created to recognize the antigen of interest. Both pAb and mAb work well for ELISA. Crude antibody preparations such as serum or ascites fluid are sometimes used for ELISA, but the impurities present may increase background. To obtain antibodies with the greatest speci- ficity, they can be affinity purified using the immobilized antigen. Either mAb or pAb may be used as the capture and detection antibodies in sandwich ELISA systems. PAbs are often used as the capture antibody to pull down as much of the antigen as possible. Then, a mAb is used as the detect- ing antibody to provide specificity. It is important to note the differences between a mAb and a pAb. MAbs are derived from a single cell line and have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. Because of this, they provide high specificity— but it is at the expense of sensitivity, since only one anti- body molecule can bind to the antigen. They are valued for their specificity, purity, and consistency, which result in lower background. On the other hand, pAbs are derived from different B-cell lines and generate a variety of responses to multi- ple epitopes with different affinities within a defined antigen. This creates a higher sensitivity because multi- ple antibodies are binding to a single antigen molecule. Polyclonal antibodies are less expensive and less time consuming to produce. Each type of antibody has pros and cons. One downside of pAbs is that they have a higher risk of nonspecific binding. But on the other hand, they show better functional affinity and cooperativity in multivalent binding across different epitopes. Therefore, they may demonstrate excellent binding overall, since they have adhered to a number of different sites on a complex immunogen or antigen. In fact, for assays requiring a broad spectrum of specificity to large molec- ular weight antigens, pAbs are the clear choice. Addition- ally, pAbs are more suitable to be used as detection antibodies. One problem is that there is a finite availabil- ity of pAbs derived from a particular source, and this makes it difficult to achieve continuity of assay perfor- mance after the original source runs out. It is important to select the highest affinity and most specific antibody available. But again, balance is the key: if taken too far, this strategy can increase the susceptibility of the assay to heterogeneity of analytes. When it comes to mAbs, the single-epitope specificity makes these antibodies more vulnerable to structural or conformational changes in the epitope. To overcome this, a cocktail of mAbs is sometimes used in an assay. In gen- eral, mAbs make excellent primary antibodies in immuno- metric immunoassays; and in competitive assays for drugs, hormones or other small analytes, mAbs are the best choice for quantitative measurement. Additionally, for assays that require good reproducibility, mAbs may be a better choice than pAbs. When determining the right balance in antibody pairs, it is important to remember that antibody functions in ELISA tests are switchable. For example, some antibod- ies are better suited in a capture role but can also per- form well in a detection role with specific coupling chemistry. Finding the correct antibody pair and choos- ing each antibody’s role is a critical early step in ELISA development. An important consideration in designing an immuno- metric ELISA is that the capture and detection antibodies must recognize two nonoverlapping epitopes in the sand- wich format. When the antigen binds to the capture anti- body, the epitope recognized by the detection antibody must not be obscured or altered. Capture and detection antibodies that do not interfere with one another and can bind simultaneously are considered a matched pair and are suitable for developing a sandwich ELISA. Preparing a “self-sandwich” ELISA assay, in which the same antibody is used for the capture and detection, can limit the dynamic range and sensitivity of the assay. Besides determining which antibody should be capture antibody and which should be detection antibody, the optimum antibody concentrations for both the capture and detection antibodies are also critical. A practical approach is to coat the ELISA plate with several dilutions of both antibodies that will be used as part of the sandwich assay. Add the analyte to be measured at high, low, and zero concentrations. Use both of the antibodies at several concentrations as a secondary antibody. Determine the Absorbance Units (AUs) that yield the maximum signal- to-noise ratio or the greatest difference between the high and low analyte concentrations with the lowest variability. As a first step, these are the conditions that could be selected for the antibodies. Antibodies play fundamental roles in determining the sensitivity and dynamic range of ELISA. This is due to the actual antibody affinity for the analyte after selection and optimization of the reagents for the antibody and antigen reaction taking place. If after attempting to develop the assay the sensitivity is still not in the desired range, differ- ent antibody pairs will need to be evaluated.
  • 9. 388 The Immunoassay Handbook CAPTURE Microtiter Plate Coating The attachment of the antibodies or antigens to the sur- face of the microtiter wells involves non-covalent bonds between the hydrophobic regions of the protein and the nonpolar plastic surface. Proteins including antibodies are often readily immobilized, but the coating efficiency varies from protein to protein. Simple non-covalent adsorption of antibodies to microtiter wells is often recommended for in-house developed ELISA since it is an easy process to use. The coating buffer must be free of any protein other than the coating protein. Understanding the interfacial interactions between the solid surface and the coated protein is profoundly impor- tant during the design of the solid phase. There are many factors to consider when coating the protein to the solid phase, namely G Surface materials. G Coating chemistry. G Antigen or antibody characteristics. G Buffers. G Incubation time. G Incubation temperature. Immobilization can alter the way antibodies and antigens function and can potentially cause the antigen to lose criti- cal epitopes. This effect is greater when protein is coated at a low concentration. However, coating at a high concen- tration may result in more aggregation. It is important to note that antibodies and antigens have different physio- chemistries (e.g., hydrophobicity, isoelectric point, glyco- sylation, and molecular weight). As a result, the coating conditions should be adjusted to each antibody or antigen. In general, protein-binding capacity is proportional to the hydrophobicity on the solid surface. A highly hydro- phobic surface may cause more structural disruption to a coated macromolecular protein compared to the same protein that is not coated. Hydrophobic coating increases the capacity, and stronger blockers may be required to mask nonspecific binding. Usually, most of a protein’s hydrophilic residues are located at the outside, and most of the hydrophobic residues orientated toward the inside. Partial denaturation of some proteins results in exposure of hydrophobic regions and ensures firmer interaction with the plastic. While conformation is highly protein specific, covalent coating may cover or alter the antigen-binding site of the antibody. Several problems arise from passive adsorption, including desorption, improper orientation, denaturation, poor immobilization efficiency, and nonspecific binding along with the target molecule. Alternatively, antibodies can be attached to a microplate through the Fc region using Pro- tein A-, G-, or A/G-coated plates, which orients them prop- erly and preserves their antigen-binding capability. Precoated plates bind selectively to the desired target proteins, minimiz- ing any contamination from other molecules that are present in the preparation. It is important to ensure that the coating solution is free of detergents because detergents often com- pete for binding and cause low and/or uneven binding. In addition, the coating buffer should contain no other proteins that might compete with the target antigens for attachment to the plastic solid phase. High purity of the antigen or anti- body to be coated will increase coating efficiency as well as assay specificity. As a rule of thumb, about 0.5–1µg of IgG may be bound per cm2 in an efficient coating process. For competitive assays, a lower coating concentration usually is chosen to ensure that the antibody is the limiting factor. The following factors need to be considered during coating: G Diffusion coefficient of the attaching molecules. G Ratio of the surface area being coated to the volume of the coating solution. G Concentration of the substance being adsorbed. G Temperature. G Time. The range of protein concentrations for coating is usually within 1–10µg/mL and with a volume of 50–100µL. How- ever, the concentration should be titrated to achieve optimum antibody binding allowing for orientation and steric effects. The coating temperature is often either 37°C for 1–3h or 4°C overnight. Increasing the tempera- ture may shorten the incubation time, but some antigens are unstable at higher temperatures. The following coating buffers are often used: G 50mM carbonate, pH 9.6, G 20mM Tris–HCl, pH 8.5, G 10mM phosphate-buffered saline, pH 7.2. It is advisable to use a buffer with a pH value 1–2 units higher than the pI value of the protein being attached, avoiding protein precipitation during the coating. The amount of 50–500ng per well has been found valid for a variety of proteins in 50µL volumes. Blocking Blocking is another essential step in ELISA, particularly when reducing the nonspecific binding of primary or sec- ondary antibodies to the solid surface of microtiter plates and nonspecific binding with low affinity from sample to solid surface. Blocking the unoccupied sites on the surface of the well can reduce the amount of nonspecific binding of proteins during subsequent steps in the assay. Therefore, it can also increase assay sensitivity and specificity. Here again, no single blocking agent is ideal for every occasion because each antibody–antigen pair has unique character- istics. A variety of blocking buffers ranging from nonfat milk to highly purified proteins have been used to block unreacted sites. Empirical testing is necessary. The proper choice of blocker for a given assay depends on the antigen itself and on the type of enzyme conjugate to be used. For example, with applications using an alkaline phosphatase (ALP) conjugate, a blocking buffer in TBS should be selected because PBS interferes with ALP. The ideal block- ing buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding. Separation and Washing The sample may contain other biological or chemical con- stituents that can interfere with the subsequent signal
  • 10. 389CHAPTER 5.1 Practical Guide to ELISA Development generation stage that need to be washed away after capture of the analyte. After incubation with the enzyme-labeled antibody, the unbound enzyme must also be removed by washing to reduce the assay nonspecific binding. Washing is one of the critical steps of ELISA. It can be easily and reproducibly carried out with inexpensive washing devices, which are manual, semiautomated or fully automated. The washing buffer usually contains 0.15mol/L NaCl and 0.05% Tween 20. The frequency of washing depends on the step. Three to five washing steps with 0.5mL of washing buffer are recommended after each incubation stage. Slamming the inverted plate onto a wad of tissues is a good way of removing any residual droplets of wash solution, and this can improve the precision of some assays. But it is important to keep the wells moist at all times between each washing step. If they dry out, the washing may not be effective, leading to high background. Signal Generation and Detection SECONDARY ANTIBODY For an in-house ELISA, it is simplest to use an off-the-shelf commercial enzyme-labeled antibody for the signal genera- tion. This is known as a secondary antibody because it is not specific for the antigen under test but for the detection antibody. The choice of secondary antibody depends upon the species of animal in which the primary antibody was raised (the host species). For example, if the primary anti- body is a rabbit antibody, the secondary antibody could be an anti-rabbit from goat, chicken, etc. but not rabbit. If a sec- ondary antibody causes high background in an assay, a sec- ondary antibody from another species may improve results. An alternative option to reduce background is to use a secondary antibody that has been pre-adsorbed to serum proteins from other species. This pre-adsorption process removes antibodies that have the potential to cross-react with serum proteins, including antibodies. Antibodies for ELISA are typically used as diluted 1/100–1/500,000 starting from a 1mg/mL stock solution. The optimal dilu- tion of a given antibody with a particular detection system must be determined experimentally. Antibody dilutions are typically made in the wash buffer containing a blocking agent. The presence of a small amount of blocking agent and detergent in the antibody diluent often helps to mini- mize background. Many labeled secondary antibodies are commercially available. Using secondary antibody, the sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary anti- body, allowing for signal amplification. However, it can be susceptible to nonspecific binding due to the presence of the secondary antibody and the extra incubation step. CHOICE OF ENZYME The most commonly used enzymes are horseradish per- oxidase (HRP) and ALP. These enzymes can be detected further with the use of colorimetric, chemiluminometric, and fluorogenic techniques. However, some of these tech- nologies require special instrumentation. An absorbance microtiter plate reader is the most practical. The results are shown through color development. A large selection of substrates is available for performing the ELISA with an enzyme conjugate. The choice of sub- strate depends upon the required sensitivity level and the instrumentation available in the individual laboratory for detection. Labeling is the process to conjugate the antigen or anti- bodies to an enzyme that can generate signal for detection in the presence of substrate and the corresponding system for measuring the captured analyte. The enzyme may be conjugated directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. However, for research purposes, direct labeling is not recommended because it is labor intensive and time consuming; it also needs special expertise. HRP is the most commonly used enzyme conjugated to antibodies in ELISA. It is a 44kDa glycoprotein with four lysine residues for conjugation to the target molecule. It can produce a colored, fluorimetric, or luminescent deriv- ative when incubated with the appropriate substrate. It also has a high turnover rate that allows generation of strong signals in a relatively short time span. HRP is active over a broad pH range with respect to its substrate: from pH 4.0 to 8.0. HRP is more stable in 0.1M citrate than 0.1M phosphate buffer. High-molar phosphate buffer can be particularly damaging to HRP at low pH. Nonionic detergents can affect the stability of the enzyme. There are a variety of substrates available for HRP, such as tetra- methylbenzidine (TMB), 3,3′-diaminobenzidine (DAB), and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). ALP is a 140 kDa protein that catalyzes the hydrolysis of phosphate groups from a substrate molecule, resulting in a colored or fluorescent product or the release of light as a byproduct. It has optimal enzymatic activity at a basic pH (pH 8–10) and can be inhibited by EDTA. Mg2+ is essen- tial for enzymatic activation. As a label for ELISA, ALP offers distinct advantages over other enzymes. The enzyme activity is not affected by exposure to antibacterial agents, such as sodium azide, which is in the formulation of many ELISA buffers. Because its reaction rate remains linear, detection sensitivity can be improved by simply allowing a reaction to proceed for longer. Nonionic detergents appear to have no effect on enzyme activation. The most common ELISA substrate for alkaline phosphatase is the chromogen p-nitrophenyl phosphate (PNPP), which is available in several formats. For example, Fischer Scien- tific provides PNPP as dry crystalline powder, single-use tablets, a stable substrate solution and as a kit containing substrate solution and diethanolamine buffer (www.fisch- ersci.com). Chemiluminescent substrates are suitable for both enzymes. Chemiluminescent substrates differ from other substrates in that the signal is a transient product of the reaction that is only present while the enzyme–substrate reaction is occurring. This is in contrast to substrates that produce a stable, colored product, which persist in the well after the enzyme–substrate reaction has termi- nated. In a chemiluminescent ELISA, the substrate is the
  • 11. 390 The Immunoassay Handbook limiting reagent in the reaction; as it is exhausted, light production decreases and eventually ceases. Therefore, a well-optimized procedure using the proper antibody dilutions will produce a reproducible output of light. If the antibody is not diluted sufficiently, too much enzyme is present, and the substrate is used up quickly, so a stable output of light will never be achieved. This is the single greatest cause of variability in chemiluminescent ELI- SAs. To avoid this problem, it is crucial to optimize the amount of antibody used for detection. Antibody suppli- ers typically suggest a dilution range for using their anti- body in an ELISA. This dilution range is often appropriate for ELISAs detected with a relatively insensitive chro- mogenic substrate, but a much greater dilution is gener- ally required for optimum performance with a sensitive chemiluminescent substrate. In competitive immunoassays, labeling an antigen may alter its epitopes and affect the antigenicity for the detec- tion antibody, but the converse can also be true and label- ing may augment the recognition. Typically this is due to bridge recognition, in which the labeling is directed to the same position that the hapten was originally conjugated to on the immunogen. The following factors need to be con- sidered when conjugating antibodies or antigens: G Size of enzyme. G Ease of conjugation. G Stability. G Cost. In general, HRP is ideal in many respects for these appli- cations because it is smaller, more stable, and less expen- sive than ALP. For both enzymes, the substrates can be made up in buffer stored frozen in well-sealed vials and then thawed for use. The substrate needs to be stable. Finally, strong acids or bases stop enzymatic activity by quickly denaturing enzymes. Sodium azide is a potent inhibitor of HRP, whereas EDTA inhibits ALP by the chelation of metal cofactors. The generation of color is controlled by the addition of a stopping reagent, which must be added at an accurately controlled volume, since photometric readings are affected if the total volume of reactants varies. Color is measured using a spectrophotometric plate reader. The product of substrate catalysis by the enzyme is measured by transmit- ting light of a specific wavelength through the solution and measuring the amount of absorption of that light. The correct filter wavelengths should be selected for spectro- photometric reading. Assay Optimization The design of ELISA is a science, because many experi- ments need to be carried out to achieve the best results with a unique set of biological, chemical, and physical ele- ments. It is also a craft, in which practitioners evolve their own sequence of activities, based on their experiences and those who teach them. But it is also an art, because the most experienced ELISA developers do not follow a fixed protocol, but view the final ELISA as a unique integrated system, transcending the many elements involved to achieve a blend of goals that is defined by the assay creator. The fundamental objectives of any ELISA development are to (1) Identify the correct antibody or antigen. (2) Create an optimum environment for assay kinetics and binding affinities. (3) Provide consistent results with maximum assay sen- sitivity and specificity. To meet these objectives, researchers need to balance many factors—all within the constraints imposed by qual- ity, cost, and time. KINETICS AND THE INCUBATION TIME Once the formulations of the reagents have been estab- lished, assay performance is optimized. By carrying out simple kinetic experiments, the relationships between incubation times and signal generation intensity are estab- lished for a range of reagent concentrations. The goal is to achieve a high signal-to-noise ratio, not simply to maxi- mize the signal. Sensitivity and precision are key goals. Practical considerations also apply, for example, the incu- bation time needs to be acceptable. Faster assays may be achieved with higher concentrations of conjugate (enzyme- labeled antigen or antibody), but the cost and availability of the conjugate are another consideration. There are usually trade-offs between desirable perfor- mance characteristics, for example, a short incubation time may result in weak signal generation, and a long incuba- tion time may favor nonspecific signal generation. The incubation time optimization often starts with observation of the impact of time and temperature on signal intensity and nonspecific binding. Adjusting incubation time can often be beneficial to reducing nonspecific binding. If this does not achieve the desired results, using more specific mAbs should result in a higher signal-to-noise ratio. Immobilization of one reactant to the solid phase may mean it takes longer to reach equilibrium than if the reac- tant was in solution. Assays with requirements for a fast turnaround time, such as cardiac markers, typically use high concentrations of conjugate to speed up the assay reaction. Cross-reactivity often decreases with incubation time and is minimal when equilibrium is reached. Increas- ing the incubation temperature may also decrease cross-reactivity. In kinetics, overall balance is important. Kinetics should be optimized in combination with other factors such as reaction volume, number of steps, conjugate mass, and matrix effects. For instance, a smaller volume may be favorable for a reaction, but a relatively larger volume would create less variation. Kinetics are also dependent on pH, ionic strength, and temperature. Ion content and pH in particular can affect assay kinetics and may affect some mAbs more than others. Another consideration: sandwich and competitive assays have different thermodynamic equilibriums. Sandwich formats generally require shorter incubation times because it is acceptable to use higher capture antibody and conju- gate concentrations. Meanwhile, competitive formats require careful consideration of reagent inputs. In addi- tion, as antibody inputs are adjusted, it is important to con- sider the high-dose hook effect in sandwich assays.
  • 12. 391CHAPTER 5.1 Practical Guide to ELISA Development BUFFERS Buffers are the backbone of ELISA. Multiple factors should be considered in buffer formulation such as the buffer system (Tris, phosphates, HEPES, or MES, etc.), ionic strength, pH, salt, detergent, proteins, blocking agents, preservatives, and other additives. Reagent formu- lation is unique to each ELISA method; and while using reagents off the shelf may shorten development time; this practice may also lead to potential problems because the reagents are not optimized. It is particularly important to consider antigen charac- teristics. In general, the use of detergents may help reduce nonspecific binding. But the proper use of a detergent depends upon the specific antigen. An antigen with epitope made up of continuous segments of polypeptide chain (lin- ear epitope) can better stand detergent and vigorous for- mulation compared to a conformational epitope, which is made up of a juxtaposition of multiple segments from dif- ferent parts of the protein sequences. In fact, the presence of a conformational epitope may limit or inhibit the use of a detergent, or high salt, and require a specific pH range. In addition, matching the surface charge plays an impor- tant role in specificity, especially for highly charged pro- tein antigens. Charge is a strong force in non-covalent antibody–antigen binding. Therefore, neutralizing the charge can help to stabilize this kind of antibody–antigen complex and lead to better recognition. Another consider- ation is antigen size. The binding site can be structurally classified into three major types: cavities, grooves, and pla- nar sites. These correspond to the size and shape of the antigen being bound. Small molecules or short peptides typically bind in a pocket or groove lying between the heavy and light chain variable regions, and there may only be contact between one to two amino acids in the antibody molecule and the epitope. As a result, small antigens may be very sensitive to subtle changes in buffer formulation or optimization. Large protein antigens bind in the planar site and may contact 15–20 amino acids in the binding site. Here, buffer optimization may result in better surface complementarity, which is also very important to the mobility, accessibility, and antigenicity of an antigen. Flex- ibility of the antigen will allow its epitope to more readily assume the best and most avid configuration in the anti- body-binding site. BLOCKING Signal-to-noise ratio is the best indicator for the correct selection of blocker(s). And as with the other steps, there is a question of balance. Over-blocking may alter or obscure the epitope for antibody binding. In addition, excessive blocker could mask antibody–antigen interac- tions, or inhibit the enzyme, and result in a reduction of the signal-to-noise ratio. REAGENT FORMULATION AND TITRATION The goals in developing an ELISA assay are G To achieve the best signal-to-noise ratio for the sensi- tivity level desired. G To be able to measure the antigen or antibody over a dynamic range. G To have a robust, reproducible assay for the sample being tested. Therefore, optimal concentrations of each assay reagent must be established empirically. The signal-to-noise ratio is the ratio of the signal level of a sample containing the target analyte to the level of noise. Noise is the stan- dard deviation (SD) of the signal when a sample from which the analyte is absent is repeatedly tested. As the sig- nal-to-noise ratio increases, the assay becomes more effec- tive at measuring small amounts of antigen. There are two approaches to increasing the signal-to- noise ratio: reduce the noise or increase the signal for a given analyte concentration. By adjusting the reagent dilu- tions, the optimum signal-to-noise ratio may be achieved empirically. One way to establish the optimal dilutions is using checkerboard titration, also called a two-dimensional serial dilution. A checkerboard titration is a single exper- iment in which the concentration of two components is varied in a way that will result in a pattern. This design permits analysis of different concentrations of the two reagents in each well to obtain the highest signal-to-noise ratio. SAMPLE VOLUME Yet another factor that needs to be taken into account is sample volume. Sample has an evident impact on reagent formulation for assay development. Ideally, sample volume should be defined by minimal interference and matrix effect. Assays vary in their susceptibility to this type of effect. The antibody–antigen reaction occurs at the solid– liquid interface, so the exact reaction volume is difficult to determine. Large sample volumes may increase assay sensi- tivity, but they may also result in higher matrix effects and lower linearity. Sample matrix effects can be minimized by using a low ratio of the sample compared to the assay reagent in the incubation. However, this reduces the signal level and potentially the signal-to-noise ratio, reducing the sensitivity of the assay. Alternatively, increasing protein and ionic concentration, as well as buffering capacity, may also mitigate matrix effects from samples. Hardware and Software INSTRUMENTATION The instrumentation to measure the signal reflecting the extent of antigen–antibody binding is determined by the conjugated enzyme and substrate combination. In ELISA methods, a soluble substrate is used to generate a signal in solution. Enzyme-labeled reagents may be detected using chromogenic, chemifluorescent, or chemiluminescent substrates, using spectrophotometer, fluorometer, or luminometer, respectively. Where the detection is colori- metric, a spectrophotometric plate reader is used. For absorbance measurement, especially with manual handling, dual-wavelength is commonly used. In dual-wave- length detection, the first measurement usually corresponds
  • 13. 392 The Immunoassay Handbook to the maximum absorbance, and the second measurement is taken at a wavelength near the baseline as a blank reference value for each well, which includes not only baseline absor- bance but also absorbance due to exogenous materials that could randomly adhere to the outside of the wells and con- tribute to imprecision. The microplate readers conveniently subtract the second absorbance reading from the first mea- surement and relate the difference to the analyte concentra- tion (Diamandis et al., 1996). The instrument used to read the output of the ELISA should be tested initially for both linearity and perfor- mance. Instrument performance should be regularly tested and recalibrated according to the manufacturer’s specifications. CALIBRATION CURVE FITTING The computerized fitting of a calibration curve is carried out using a commercial product. A printout of the curve indicates whether there are any biases between the com- puted curve and the calibrator signal levels. This subject is well covered in the chapters CALIBRATION CURVE FITTING and IMMUNOASSAY TROUBLESHOOTING GUIDE. Assay Management STANDARDIZATION AND CALIBRATION Known concentrations of analyte are used to provide cali- bration curves against which unknown sample concentra- tions can be ascertained. See the chapters STANDARDIZATION AND CALIBRATION and IMMUNOASSAY TROUBLESHOOTING GUIDE. QUALITY CONTROL AND VALIDATION The purpose of quality control is to independently verify the reproducibility and, if there is a standard, the accuracy, of the results obtained using the ELISA method. Control samples can be from patients or made by spiking and pool- ing. Ideally, the analyte concentration in the control sam- ples should have been determined by another validated method. Spiked controls are created by adding a known concentration of the standard analyte into the usual sample matrix and used in each experiment to track method per- formance. Spiked controls can be used to determine assay performance based on calculating the percent dose recov- ery. The following features need to be evaluated in choos- ing an antigen for quality controls: G Purity. G Reactivity. G Stability. G Traceability. ELISA intended for research use needs to meet the required specifications for method performance. Before designing the ELISA method, it is advisable to define the achievable specifications. By setting the bar for the new method too high, there is a risk that development will be thwarted rather than encouraged. Major parameters to evaluate during the validation are G Sensitivity and specificity. G Imprecision and repeatability. G Linearity and stability. G Cross-reactivity and interference. ELISA Tips and Troubleshooting FACTORS AFFECTING ELISA RESULTS G Temperature: temperature variation will affect anti- body-binding affinities. Variation is most likely between the corners and center of a microtiter plate. A stack of plates is more vulnerable, between the corners of the top and bottom plates and the center of the stack. Test for temperature effects by assaying repli- cates of one control or sample pool across an entire plate. Calculate the mean and SD of the optical densi- ties (ODs) from all the wells and then place each well signal level in one of the following groups: ≥2 SD, −2 to −1 SD, −1 to 0 SD, 0 to +1 SD, 1–2 SD, and >2 SD. Plot the distribution of signal levels across the plate by marking each well position with −−− (≥2 SD), −− (−2 to −1 SD), − (−1 to 0 SD), + (0 to +1 SD), ++ (1–2 SD), and +++ (>2 SD). Effects due to temperature gradients in the incubator will show up clearly. If patterns show up either increase the incubation time or improve the plate incubator. Temperature effects have characteris- tic patterns, usually with the greatest difference from the center to the corners. This same test may also show up other characteristic patterns, such as manifold effects affecting the efficiency of plate washers (from the center line to the sides) or drift (from the first to the last well). G Time: accuracy of time is a critical matter for a con- sistent testing result. Therefore, it is recommended to always use the same procedure and follow the same order for addition of reagents, taking the same (short) time for pipetting across the plate. Time effects show up as drift from the first to the last well if a sample pool is assayed in every well. Drift can be reduced by increasing the incubation time or improv- ing the consistency of pipetting times for consecutive stages. G Movement: shaking during incubation is preferable. Plate rotation is better than leaving plates stationary. It eliminates viscosity effects and reduces the likelihood of temperature gradients. ELISA TIPS G Receive good training. G Follow the manufacturer’s directions. G Be careful in sample preparation and avoid samples with lipemia, hemolysis, and particulates. G Pipette with accuracy. 1. Avoid leaving the pipette on the side of the plate. 2. Avoid frothing on addition of samples. 3. Keep the same order and pace for pipetting.
  • 14. 393CHAPTER 5.1 Practical Guide to ELISA Development 4. Finish pipetting one plate within 2 min, one plate at a time. G Be careful in handling conjugates and make fresh work- ing solutions. G Be careful in handling positive samples; do not cause cross-contamination of negative wells or samples. G Carry out washing thoroughly, especially where ana- lytes are in cell lysates, or require extraction using reagents containing SDS or other denaturing reagents that may interfere with the assay. Wash with PBS thoroughly. G Make sure the plate reading is at the correct wavelength. G Use good quality water. COMMON TROUBLESHOOTING G Low ODs or false-negative results 1. Pipette function. 2. Not freshly coated plates. 3. Incomplete reagent mixing. 4. Short incubation. 5. Low temperature. 6. Incorrect plate reader filters. 7. Wrong buffers. G Increased CVs for duplicates. 1. Poor washing. 2. Alignment issues causing carryover. G No color development. 1. Incorrectly diluted or made conjugate. 2. Wrong order of reagent additions. 3. Wrong reagents or stop solution used as reagent. 4. Missed or mistaken pipetting. 5. Poor repeatability: technical accuracy, pipet- ting too fast, carryover between wells, inconsis- tent reagents, time variability, temperature variability, contamination, wrong filter, sample issue. Conclusions The ELISA method provides an ideal tool for a wide range of studies in many biological and medical laboratories. The method has high sample handling capacity, reliable analytical performance, and ease of use. It can be manually performed or run on automatic or semiautomatic plat- forms. ELISA technology depends on complexity as well as flexibility in the reagents rather than in instrumentation alone, therefore, the design of the assay is very important. However, the technical simplicity of ELISA belies the fairly complex course of ELISA development. Selecting key raw materials, optimizing the reagent formulation, and defining the processes for consistently making reagents are critical factors for developing ELISA methods. At each step, there are considerations to be made, trade-offs to consider, a balance to be achieved. And it is essential to find this balance within each step, as well as across the entire design process as a whole. It is also important to keep in mind that there is no “correct” combination of materials and conditions; the right balance depends on the specific needs that have been defined for the assay system. Ultimately, to view the development process as an inter- related system rather than many individual factors helps establish optimal conditions for assay. From this perspec- tive, the system balance can be adjusted as a whole, giving that there is no absolute, only relative. References and Further Reading Diamandis, E.P., Theodore, K.C. and Mohammad, J.K. (eds). Immunoassay, (Christopoulos, Academic Press, 1996). Ekins, R. Immunoassays: recent developments and future directions. Nucl. Med. Biol. 21, 495–521 (1994). Goldblatt, D., Van Etten, L., Van Milligen, F.J., Aalberse, R.C. and Turner, M.W. The role of pH in modified ELISA procedures used for the estimation of func- tional antibody affinity. J. Immunol. Methods 166, 281–285 (1993). Greenwood, N.P., Ovsyannikova, I.G., Vierkant, R.A., O’Byrne, M.M. and Poland, G.A. A qualitative and quantitative comparison of two rubella virus-specific IgG antibody immunoassays. Viral Immunol. 23, 353–357 (2010). Ivanov, V.S., Suvorova, Z.K., Tchikin, L.D., Kozhich, A.T. and Ivanov, V.T. Effective method for synthetic peptide immobilization that increases the sensi- tivity and specificity of ELISA procedures. J. Immunol. Methods 153, 229–233 (1992). Jeney, C., Dobay, O., Lengyel, A., Adám, E. and Nász, I. Taguchi optimisation of ELISA procedures. J. Immunol. Methods 223, 137–146 (1999). Martens, C., Bakker, A., Rodriguez, A., Mortensen, R.B. and Barrett, R.W. A generic particle-based nonradioactive homogeneous multiplex method for high-throughput screening using microvolume fluorimetry. Anal. Biochem. 273, 20–31 (1999). Rees, A.R., Staunton, D., Webster, D.M., Searle, S.J., Henry, A.H. and Pedersen, J.T. Antibody design: beyond the natural limits. Trends Biotechnol. 12, 199–206 (1994). Ruths, S., Driedijk, P.C., Weening, R.S. and Out, T.A. ELISA procedures for the measurement of IgG subclass antibodies to bacterial antigens. J. Immunol. Methods 140, 67–78 (1991). Steinitz, M. and Baraz, L. A rapid method for estimating the binding of ligands to ELISA microwells. J. Immunol. Methods 238, 143–150 (2000). Stenman, U.H. Improving immunoassay performance by antibody engineering. Clin. Chem. 51, 801–802 (2005). Stockwell, B.R., Haggarty, S.J. and Schreiber, S.L. High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post- translational modifications. Chem. Biol. 6, 71–83 (1999). Swartzman, E.E., Miraglia, S.J., Mellentin-Mitchelotti, J., Evangelista, L. and Pau-Miay, Y. A homogeneous multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology. Anal. Biochem. 271, 143–151 (1999). Székács, A., Le, H.T.M., Szurdoki, F. and Hammock, B. Optimization and valida- tion of an enzyme immunoassay for the insect growth regulator fenoxycarb. Analytica. Chimica. Acta 487, 15–29 (2003). Wierdal, D. and Zuckermanm, L. Recommendations for the design and optimiza- tion of immunoassays used in the detection of host antibodies against biotech- nology products. J. Immunol. Methods 289, 1–16 (2004). Zuck, P., Lao, Z., Skwish, S., Glickman, J.F., Yang, K., Burbaum, J. and Inglese, J. Ligand–receptor binding measured by laser scanning imaging. PNAS 96, 11122–11127 (1999). http://www.brendan.com/